Immunoplate-MALDI MS platform and assays

PROJECT SUMMARY The goal of this proposal is to develop simple, robust and cost-effective Mass Spectrometry (MS)-based protein assays and platform by combining the best aspects of enzymatic immunoassays – plate based protein immunoaffinity capture, with the straightforward MS detection offered by MALDI-TOF MS. These immunoplate- MALDI MS assays are improved version of the enzymatic immunoassays wherein the secondary reporter antibody detection step is replaced with MS detection, enabling unambiguous protein detection and identification through specific and accurate measurements of its mass. The impetus for the development of the new platform and assays is the ability to detect and study human proteoforms - the different molecular forms in which the protein product of a single gene exist. Mass spectrometry is uniquely capable of providing measurements of individual proteoforms masses that vary from the original protein sequence. These proteoforms are best detected intact, which is readily achieved by MALDI- TOF MS. A MALDI mass spectrum of an affinity–retrieved protein displays all proteoforms captured by an antibody-coated immunoplate well. Relative ratios of the proteoforms can be determined from their signals in the mass spectra, and reported as % abundance. The new platform and assays will meet the three key enabling factors for MS protein assays: content, cost, and simplicity. In this Phase I research we will demonstrate the platform via proof-of-principle immunoplate-MALDI MS assays for six protein biomarkers that exhibit proteoforms: apolipoprotein E, transferrin, transthyretin, cystatin C, retinol binding protein, and B-type natriuretic peptide. These protein biomarkers span a wide spectrum of concentrations and molecular weights, which will enable us to test the platform range, capabilities and limitations. Immunoaffinity isolation of the proteins from human plasma samples will be achieved utilizing antibody-coated 96-well plates, after which the captured proteins and their proteoforms will be eluted and spotted on a MALDI target for MS analyses. Experimental conditions will be tested for the antibody adsorption, human plasma assaying, and elution onto MALDI targets. The number of steps will be kept to a minimum, yet enough to obtain a satisfactory performance at the lowest cost possible. The reproducibility of the new assays will be evaluated, and the assays will be benchmarked. Our goal is to develop the immunoplate-MALDI MS platform for the general RandD market. Assay designed on this platform will be cost-effective and simple, and can readily be adopted by laboratories with existing MALDI MS instrumentation. The new platform and assays can be used in proteoform discovery efforts, as well as larger clinical validation studies to delineate the clinical correlations of specific proteoforms.